Plinabulin compositions

ABSTRACT

Disclosed herein are plinabulin polymorphs, compositions, their use and preparation as therapeutic agents. In particular, some embodiments relate to plinabulin monohydrate in a crystalline form.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/741,635 filed on Jan. 3, 2018, issued as U.S. Pat. No. 10,155,748 onDec. 18, 2018, which is the U.S. National Phase of InternationalApplication No. PCT/US2016/041773 entitled PLINABULIN COMPOSITIONS,filed Jul. 11, 2016 and published on Jan. 19, 2017 as WO 2017/011399,which claims the benefit of U.S. Provisional Application No. 62/191,990,filed Jul. 13, 2015, the disclosure of which are incorporated herein byreference in their entireties.

BACKGROUND Field

The present invention relates to the fields of chemistry and medicine.More particularly, the present invention relates to forms andcompositions of plinabulin and their preparation.

Description of the Related Art

Plinabulin is a synthetic analog of diketopiperazine phenylahistin(halimide) discovered from marine and terrestrial Aspergillus sp.Plinabulin is structurally different from colchicine and itscombretastatin-like analogs (eg, fosbretabulin) and binds at or near thecolchicine binding site on tubulin monomers. Previous studies showedthat plinabulin induced vascular endothelial cell tubulindepolymerization and monolayer permeability at low concentrationscompared with colchicine and that it induced apoptosis in Jurkatleukemia cells. Studies of plinabulin as a single agent in patients withadvanced malignancies (lung, prostate, and colon cancers) showed afavorable pharmacokinetic, pharmacodynamics, and safety profile.

SUMMARY OF THE INVENTION

Some embodiments relate to a plinabulin monohydrate.

Other embodiments relate to a plinabulin monohydrate in crystallineform.

Some embodiments relate to a plinabulin composition having more thanabout 90% by weight of plinabulin, based on the total weight of thecomposition.

Other embodiments relate to a plinabulin composition having more thanabout 99% by weight of plinabulin, based on the total weight ofmolecules in the composition other than water, dimethylformamide,ethanol, ethyl acetate, methanol, toluene, and acetic acid.

Some embodiments relate to a plinabulin composition, containingplinabulin and no more than about 1.9% by weight of impurities, based onthe total weight of the composition other than water.

Other embodiments relate to a plinabulin composition, containingplinabulin and no more than about 1% by weight of impurities other thansolvent molecules, based on the total weight of non-solvent molecules inthe composition.

Some embodiments relate to a plinabulin composition having plinabulinand no more than about 1% by weight of impurities, based on a HPLCanalysis.

Some embodiments relate to a process of preparing a plinabulinmonohydrate or plinabulin composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an X-ray powder diffraction (XRPD) pattern of the crystallineform of plinabulin monohydrate.

FIG. 2 shows a polarized light microscopy (PLM) image of a samplecontaining the crystalline form of plinabulin monohydrate.

FIG. 3A shows the thermo gravimetric (TGA) of the crystalline form ofplinabulin monohydrate (Form 1); FIG. 3B shows the digital scanningcalorimetry (DSC) analysis results of the crystalline form of plinabulinmonohydrate (Form 1); and FIG. 3C shows the Dynamic Vapor Sorption (DVS)isotherm plot.

FIG. 4 is an XRPD pattern of the crystalline form 2.

FIG. 5 shows a PLM image of a sample containing the crystalline form 2.

FIG. 6A shows the TGA analysis of the crystalline form 2 and FIG. 6Bshows the DSC analysis results of the crystalline form 2.

FIG. 7 is an XRPD pattern of the crystalline form 3.

FIG. 8 shows a PLM image of a sample containing the crystalline form 3.

FIG. 9A shows the TGA analysis of the crystalline form 3; and FIG. 9Bshows the DSC analysis results of the crystalline form 3.

FIG. 10 is an XRPD pattern of the crystalline form 4.

FIG. 11 shows a PLM image of a sample containing the crystalline form 4.

FIG. 12A shows the TGA analysis of the crystalline form 4; and FIG. 12Bshows the digital scanning calorimetry (DSC) analysis results of thecrystalline form 4.

FIG. 13 is an XRPD pattern of the crystalline form 5.

FIG. 14 shows the TGA analysis of the crystalline form 5

FIG. 15 shows the DSC analysis results of the crystalline form 5.

FIG. 16 is an XRPD pattern of the crystalline form 6.

FIG. 17 shows the TGA analysis of the crystalline form 6

FIG. 18 shows the DSC analysis results of the crystalline form 6.

FIG. 19 is an XRPD pattern of the crystalline form 7.

FIG. 20 shows the TGA of the crystalline form 7

FIG. 21 shows the DSC analysis results of the crystalline form 7.

FIG. 22 is an XRPD pattern of the crystalline form 8.

FIG. 23 shows the TGA of the crystalline form 8.

FIG. 24 shows the DSC analysis results of the crystalline form 8.

FIG. 25 is an XRPD pattern of the crystalline form 9.

FIG. 26 shows the TGA analysis of the crystalline form 9

FIG. 27 shows the DSC analysis results of the crystalline form 9.

FIG. 28 shows the inter-conversion of plinabulin polymorph forms.

FIG. 29 is a flow diagram of preparing the plinabulin monohydratecomposition.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Plinabulin,(3Z,6Z)-3-Benzylidene-6-{[5-(2-methyl-2-propanyl)-1H-imidazol-4-yl]methylene}-2,5-piperazinedione,is a synthetic analog of the natural compound phenylahistin. Plinabulincan be readily prepared according to methods and procedures detailed inU.S. Pat. Nos. 7,064,201 and 7,919,497, which are incorporated herein byreference in their entireties. Some embodiments relate to polymorphs andsolvates (e.g., hydrates) of plinabulin and pharmaceutical compositionscomprising the same. Some embodiments include methods of preparation andmethods of treatment. In particular, some embodiments relate to aplinabulin monohydrate.

Plinabulin Monohydrate

Plinabulin monohydrate (Form 1) is stable crystalline form ofplinabulin. The X-ray powder diffraction (PXRD) pattern of plinabulinmonohydrate (Form 1) is substantially the same as shown in FIG. 1, withcorresponding tabulated peak data shown in Table 1.

TABLE 1 Peak Data of X-ray powder diffraction (PXRD) pattern ofplinabulin monohydrate (Form 1) Angle (2-θ) Intensity (%) D value(angstrom) 8.14 100.0 10.854 11.16 6.2 7.923 13.08 19.7 6.764 13.91 4.96.363 14.13 5.3 6.263 14.83 6.2 5.969 15.50 5.2 5.714 16.06 8.1 5.51516.29 13.4 5.437 17.64 7.2 5.023 18.47 4.9 4.799 19.17 6.8 4.627 19.356.2 4.583 19.79 8.9 4.482 20.88 5.6 4.251 22.42 6.2 3.963 22.87 8.43.886 23.87 13.4 3.726 24.23 14.4 3.670 24.53 17.0 3.626 25.38 8.2 3.50626.59 10.8 3.350 27.19 4.8 3.277 27.44 5.5 3.248 27.95 4.8 3.190 28.905.0 3.087 29.34 9.5 3.041

In some embodiments, the plinabulin monohydrate (Form 1) describedherein includes a crystalline form exhibiting an X-ray powderdiffraction pattern comprising at least three characteristic peaksselected from the group consisting of peaks at approximately 8.1°,13.1°, 16.3°, 23.9°, 24.2°, 24.5°, and 26.6° 2θ. In some embodiments,the plinabulin monohydrate (Form 1) described herein includes acrystalline form exhibiting an X-ray powder diffraction patterncomprising at least peaks at approximately 8.1°, 13.1°, 16.3°, 23.9°,24.2°, 24.5°, and 26.6° 2θ. In some embodiments, the plinabulinmonohydrate (Form 1) described herein includes a crystalline formexhibiting an X-ray powder diffraction pattern comprising at least peaksat approximately 8.1°, 13.1°, 16.1°, 16.3°, 19.8°, 22.9°, 23.9°, 24.2°,24.5°, 26.6°, and 29.3° 2θ.

As is well understood in the art, because of the experimentalvariability when X-ray diffraction patterns are measured on differentinstruments, the peak positions are assumed to be equal if the two theta(2θ) values agree to within 0.2° (i.e., ±0.2°). For example, the UnitedStates Pharmacopeia states that if the angular setting of the 10strongest diffraction peaks agree to within ±0.2° with that of areference material, and the relative intensities of the peaks do notvary by more than 20%, the identity is confirmed. Accordingly, peakpositions within 0.2° of the positions recited herein are assumed to beidentical. Unless otherwise indicated, all X-ray diffraction anglesrecited herein are based on a copper K-alpha source.

FIG. 3B shows digital scanning calorimetry (DSC) analysis results of thecrystalline form of the plinabulin monohydrate (Form 1). As shown inFIG. 3B, the crystalline form of the plinabulin monohydrate (Form 1) hasa melting point of about 267° C.; the crystalline form of the plinabulinmonohydrate (Form 1) has a differential scanning calorimetry thermogramwith endothermic peaks at about 141° C. and about 267° C.

The crystalline form of the plinabulin monohydrate (Form 1) is morestable than the other polymorph forms. The plinabulin monohydrate(Form 1) can remain stable during the DVS and drying tests as comparedto other polymorph forms, which may show weight change and degradationduring the tests.

Plinabulin Composition

Some embodiments relate to a plinabulin composition that includes morethan about 50% by weight of the plinabulin monohydrate (Form 1)described herein, based on the total weight of the composition. In someembodiments, the plinabulin composition includes more than about 75% ofthe plinabulin monohydrate (Form 1) described herein. In someembodiments, the plinabulin composition includes more than about 90% ofthe plinabulin monohydrate described herein. In some embodiments, theplinabulin composition includes more than about 95% of the plinabulinmonohydrate described herein. In some embodiments, the plinabulincomposition includes more than about 98% of the plinabulin monohydratedescribed herein. In some embodiments, the plinabulin compositionincludes more than about 99% of the plinabulin monohydrate describedherein. In some embodiments, the plinabulin composition includes theplinabulin monohydrate described herein in the range of about 50% toabout 99%, about 60% to about 99%, about 70% to about 99%, about 80% toabout 99%, about 90% to about 99%, about 95% to about 99%, or about97.5% to about 99%, based on the total weight of the composition. Theremaining portion of the plinabulin composition may be other forms ofplinabulin and/or other chemical entities.

Some embodiments relate to a plinabulin composition with a high purity.In particularly, some embodiments relate to a plinabulin compositionhaving more than about 90% by weight of plinabulin, based on the totalweight of the composition. In some embodiments, the plinabulincomposition includes more than about 92% of the plinabulin compound. Insome embodiments, the plinabulin composition includes more than 80%,85%, 86%, 87%, 88%, 89%, 90%, 91,%, 92%, 93%, 94%, 95%, 96%, 96.5%, 96%,98%, 99%, or 99.6% of the plinabulin compound. In some embodiments, theplinabulin composition includes more than about 99% by weight ofplinabulin, based on the total weight of non-solvent molecules in thecomposition. In some embodiments, the plinabulin composition includesmore than about 96%, 97%, 98%, 99%, or 99.6% by weight of plinabulin,based on the total weight of non-solvent molecules in the composition.In some embodiments, the solvent can be water, dimethylformamide,ethanol, ethyl acetate, methanol, toluene, and acetic acid. In someembodiments, the plinabulin in the high-purity composition is present,at least in part, in plinabulin monohydrate as described above.

In some embodiments, the plinabulin composition includes more than about99% by weight of a plinabulin, based on the total weight of molecules inthe composition other than water, dimethylformamide, ethanol, ethylacetate, methanol, toluene, and acetic acid. In some embodiments, theplinabulin composition includes more than about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91,%, 92%, 93%, 94%, 95%, 96%, 96.5%, 96%, 98%, 99%, or 99.6%by weight of a plinabulin, based on the total weight of molecules in thecomposition other than water, dimethylformamide, ethanol, ethyl acetate,methanol, toluene, and acetic acid. In some embodiments, the plinabulinin the composition is present, at least in part, in plinabulinmonohydrate as described above.

In some embodiments, the plinabulin composition includes more than about99% by weight of a plinabulin, based on a HPLC analysis. In someembodiments, the plinabulin composition includes more than about 80%,85%, 86%, 87%, 88%, 89%, 90%, 91,%, 92%, 93%, 94%, 95%, 96%, 96.5%, 96%,98%, 99%, or 99.6% by weight of a plinabulin, based on a HPLC analysis.In some embodiments, the plinabulin in the composition is present, atleast in part, in plinabulin monohydrate as described above.

Some embodiments relate to a plinabulin composition with low levels ofimpurities. The term “impurity” as used herein refers to one or morecomponents of the composition that is different from plinabulin andwater. In some embodiments, the impurity can include one or morechemical compounds introduced during the synthesis of plinabulin. Insome embodiments, the impurity can include dimethylformamide, ethanol,ethyl acetate, methanol, toluene, acetic acid, and other residualsolvent.

In some embodiments, the plinabulin composition includes no more thanabout 1% by weight of impurities, based on the total weight of thecomposition. In some embodiments, the plinabulin composition includes nomore than about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%,3%, 2%, 1%, 0.97%, 0.9%, 0.8%, 0.6%, 0.4% or 0.2% by weight ofimpurities, based on the total weight of the composition. In someembodiments, the plinabulin composition includes no more than about 1%by weight of impurities, based on the total weight of non-solventmolecules in the composition. In some embodiments, the plinabulincomposition includes no more than about 15%, 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.97%, 0.9%, 0.8%, 0.6%, 0.4% or0.2% by weight of impurities, based on the total weight of non-solventmolecules in the composition.

In some embodiments, the plinabulin composition includes no more thanabout 1.9% by weight of impurities, based on the total weight of thecomposition other than water. In some embodiments, the plinabulincomposition includes no more than about 15%, 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.97%, 0.9%, 0.8%, 0.6%, 0.4% or0.2% by weight of impurities, based on the total weight of thecomposition other than water. In some embodiments, the plinabulincomposition includes no more than about 1% by weight of impurities,based on the total weight of non-solvent molecules in the compositionother than water. In some embodiments, the plinabulin compositionincludes no more than about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, 0.97%, 0.9%, 0.8%, 0.6%, 0.4% or 0.2% by weightof impurities, based on the total weight of non-solvent molecules in thecomposition other than water.

In some embodiments, the plinabulin composition includes no more thanabout more than about 0.9% by weight of impurities other thandimethylformamide, ethanol, ethyl acetate, methanol, toluene, and aceticacid, based on the total weight of molecules in the composition otherthan water, dimethylformamide, ethanol, ethyl acetate, methanol,toluene, and acetic acid. In some embodiments, the plinabulincomposition includes no more than about more than about 5%, 4%, 3%, 2%,1%, 0.97%, 0.9%, 0.8%, 0.6%, 0.4% or 0.2% by weight of impurities otherthan dimethylformamide, ethanol, ethyl acetate, methanol, toluene, andacetic acid, based on the total weight of molecules in the compositionother than water, dimethylformamide, ethanol, ethyl acetate, methanol,toluene, and acetic acid.

In some embodiments, the plinabulin composition includes no more thanabout more than about 1% by weight of impurities, based on a HPLCanalysis. In some embodiments, the plinabulin composition includes nomore than about more than about 5%, 4%, 3%, 2%, 1%, 0.97%, 0.9%, 0.8%,0.6%, 0.4% or 0.2% by weight of impurities, based on a HPLC analysis.

Method of Preparation

Some embodiments relate to a process of preparing the plinabulinmonohydrate or the plinabulin composition described herein, the methodincluding: combining plinabulin and a first solvent system to form afirst mixture, heating the first mixture to a temperature in the rangeof about 50° C. to 90° C., and cooling the first mixture to form a firstprecipitate.

In some embodiments, the process further includes filtering prior tocooling the first mixture. In some embodiments, the process furtherincludes adding water to the first mixture prior to heating.

In some embodiments, the process described herein further includesfiltering the first precipitate. In some embodiments, the processdescribed herein further includes washing the first precipitate.

In some embodiments, the first solvent system can be water, alcohol, ora mixture of water and alcohol.

In some embodiments, the alcohol is selected from methanol, ethanol,isopropyl alcohol, tert-butyl alcohol and n-butyl alcohol; or mixturethereof.

In some embodiments, the alcohol is ethanol.

In some embodiments, heating the first mixture comprises refluxing thefirst mixture.

In some embodiments, the first mixture is heated to about 70° C. to 78°C. In some embodiments, the first mixture is heated to about 60° C. to90° C. In some embodiments, the first mixture is heated to about 60° C.to 80° C. In some embodiments, the first mixture is heated to theboiling point of ethanol.

In some embodiments, the process described herein further includesmaintaining the first mixture at a refluxing temperature for about 1hour prior to cooling the first mixture.

In some embodiments, heating the first mixture includes heating thefirst mixture to at least 65° C., and wherein cooling the first mixtureincludes cooling the first mixture to about 50° C. to 60° C.

In some embodiments, the cooling of the first mixture includes addingwater to the first mixture to produce the first precipitate.

In some embodiments, the cooling of the first mixture includes stirringthe first mixture for at least 4 hours.

In some embodiments, the process described herein further includesanalyzing the first precipitate to determine the plinabulin compositionin the first precipitate.

In some embodiments, the process described herein further includescombining the first precipitate and a second solvent to form a secondmixture and heating the second mixture to a temperature in the range ofabout 50° C. to 90° C.; cooling the second mixture to form a secondprecipitate; and filtering the second precipitate and washing the secondprecipitate.

In some embodiments, the second solvent is water, alcohol, or a mixtureof water and alcohol.

In some embodiments, the alcohol is selected from methanol, ethanol,isopropyl alcohol, tert-butyl alcohol and n-butyl alcohol; or mixturethereof. In some embodiments, the alcohol is ethanol.

In some embodiments, heating the second mixture comprises refluxing thesecond mixture.

In some embodiments, the second mixture is heated to about 70° C. to 78°C.

In some embodiments, the process described herein includes maintainingthe second mixture at a refluxing temperature for about 1 hour prior tocooling the second mixture.

In some embodiments, cooling the second mixture comprises cooling thefirst mixture to about 15° C. to 30° C.

In some embodiments, the cooling of the second mixture comprises addingwater to the second mixture to produce the second precipitate.

In some embodiments, the cooling of the second mixture comprisesstirring the second mixture for at least 4 hours.

In some embodiments, the first precipitate is washed with an alcohol andthe washed alcohol is collected and added to the second mixture prior tothe heating.

In some embodiments, the process described herein includes drying thesecond precipitate

In some embodiments, the process described herein includes analyzing thesecond precipitate to determine the plinabulin composition in the secondprecipitate.

In some embodiments, the combining, cooling, and filtering steps arerepeated one or more times based on the plinabulin composition in thesecond precipitate.

Some embodiments relate to a process of preparing the plinabulinmonohydrate or the plinabulin composition, wherein the process includesmixing plinabulin, ethanol, and water to form a mixture. In someembodiments, the process includes the mixture.

In some embodiments, the volume ratio of the ethanol to water is about95:5. In some embodiments, the volume ratio of the ethanol to water isabout 85:15, 90:10, 95:5, 97.5:2.5, or 99:1. In some embodiments, thevolume ratio of the ethanol to water is about 15, 16, 17, 18, 19, 20,21, 22, 23, 24, or 25.

In some embodiments, the mixture of plinabulin and the solvent system isstirred for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20hours. In some embodiments, the mixture is stirred for at least 2 hours.

In some embodiments, the mixing or stirring is performed at atemperature in the range of about 10° C. to about 50° C.; about 20° C.to about 40° C.; about 25° C. to about 35° C., or about 28° C. to about32° C. In some embodiments, the mixing of plinabulin, ethanol and waterto form the mixture or stirring of the mixture is performed at about 20°C., 25° C., 30° C., 35° C., or 40° C.

FIG. 29 shows a block diagram of one method for producing the plinabulinmonohydrate form composition. As shown in FIG. 29, the plinabulincompound and ethanol are added to a reaction flask, and the mixture isthen heated to 70-78° C. and mixed at this temperature for about 1 hour.Additional ethanol can be added if needed. The flask is then cooled toabout 50-60° C., the mixture is filtered, and ethanol is used as a rinsesolvent. The filtered solution and the rinse solution are combined andwater is added to about 10% of the combined solution. The solution isthen heated to 70-78° C. and mixed at this temperature for about 1 hour.A sample is taken for XRPD analysis, and then the solution is cooled to20±5° C. and added with water. The batch is filtered and water is usedas a rinse. The filtered product is washed with water and thentransferred to the drying trays to dry at 40-50° C. for about 24 hoursor longer until it reaches the required ethanol and water contentlevels.

Plinabulin Crystalline Form 2

Some embodiments relate to a crystalline Form 2 of plinabulin and itsprocess of preparation. While not being bound by any particular theory,it is believed that Form 2 is a plinabulin isopropyl alcohol (IPA))solvate.

In some embodiments, crystalline Form 2 of plinabulin has substantiallythe same XRPD pattern as depicted in FIG. 4, with correspondingtabulated peak data shown in Table 2.

TABLE 2 Peak Data of PXRD pattern of crystalline Form 2 Angle, 2θ dspacing Intensity, % 9.93 8.90 100 13.25 6.68 39.6 15.96 5.55 7.9 16.285.44 3.7 16.69 5.31 2.2 18.47 4.80 5.5 18.68 4.75 9 19.45 4.56 28.919.90 4.46 4.2 20.53 4.32 3.1 22.71 3.91 14 23.51 3.78 6 24.44 3.64 225.12 3.54 6.7 25.45 3.50 2.6 26.06 3.42 1.4 26.19 3.40 1.9 27.69 3.225.9 28.04 3.18 2.3 29.27 3.05 4

The crystalline Form 2 may further be characterized by a DSC thermogramsubstantially the same as that depicted in FIG. 6B. As shown in FIG. 6B,the crystalline Form 2 has a melting point of about 267° C.; thecrystalline form of plinabulin Form 2 has a differential scanningcalorimetry thermogram with endothermic peaks at about 113° C. and about267° C.

Plinabulin Crystalline Form 3

Some embodiments relate to a crystalline Form 3 of plinabulin and itsprocess of preparation. While not being bound by any particular theory,it is believed that Form 3 is an anhydrous form of plinabulin.

In some embodiments, crystalline Form 3 of plinabulin has substantiallythe same XRPD pattern as depicted in FIG. 7, with correspondingtabulated peak data shown in Table 3.

TABLE 3 Peak Data of PXRD pattern of crystalline Form 3 Angle, 2θ dspacing Intensity, % 7.75 11.40 4.3 8.84 9.99 17.8 9.97 8.86 7.9 10.218.66 59.2 10.86 8.14 13.9 11.99 7.38 5.2 12.87 6.87 3.2 13.69 6.46 10.315.87 5.58 32.7 16.15 5.48 11.7 16.71 5.30 41.9 17.54 5.05 23.9 17.735.00 25.8 17.86 4.96 8 18.19 4.87 31.4 18.59 4.77 3.9 19.15 4.63 2.319.50 4.55 42.4 20.06 4.42 7.5 20.52 4.32 100 21.58 4.11 14.2 22.08 4.0219.4 22.92 3.88 10.9 23.35 3.81 25.3 24.54 3.63 20.3 24.80 3.59 2.825.10 3.55 6.2 25.34 3.51 3.8 25.89 3.44 4.5 26.53 3.36 10.2 27.35 3.262.7 27.65 3.22 9.4 27.93 3.19 11.2 29.13 3.06 11.3 29.54 3.02 3.1 29.8129.81 29.81

The crystalline Form 3 may further be characterized by a DSC thermogramsubstantially the same as that depicted in FIG. 9B. As shown in FIG. 9B,the crystalline Form 3 has a melting point of about 264° C.; thecrystalline Form 3 has a differential scanning calorimetry thermogramwith endothermic peak at about 264° C.

Plinabulin Crystalline Form 4

Some embodiments relate to a crystalline Form 4 of plinabulin and itsprocess of preparation. While not being bound by any particular theory,it is believed that Form 4 is a plinabulin methanol solvate.

In some embodiments, crystalline Form 4 of plinabulin has substantiallythe same XRPD pattern as depicted in FIG. 10, with correspondingtabulated peak data shown in Table 4.

TABLE 4 Peak Data of PXRD pattern of crystalline Form 4 Angle, 2θ dspacing Intensity, % 7.41 11.93 7.9 7.71 11.45 100 9.06 9.76 38.6 12.167.28 10.2 12.50 7.07 5.6 12.74 6.94 2.2 15.44 5.73 28.2 15.70 5.64 7.216.27 5.44 10.1 16.72 5.30 17.4 17.33 5.11 4.6 17.56 5.05 10.5 18.134.89 14.6 18.79 4.72 17.5 19.39 4.57 3.5 20.03 4.43 9.6 21.53 4.12 723.32 3.81 12.4 23.90 3.72 33.5 24.42 3.64 26.2 25.69 3.47 5.3 27.083.29 3.3 28.15 3.17 4.4 28.90 3.09 9.6

The crystalline Form 4 may further be characterized by a DSC thermogramsubstantially the same as that depicted in FIG. 12B. As shown in FIG.12B, the crystalline Form 4 has a melting point of about 267° C.; thecrystalline Form 4 has a differential scanning calorimetry thermogramwith endothermic peaks at about 113° C. and at about 264° C.

Plinabulin Crystalline Form 5

Some embodiments relate to a crystalline Form 5 of plinabulin and itsprocess of preparation.

In some embodiments, crystalline Form 5 of plinabulin has substantiallythe same XRPD pattern as depicted in FIG. 13, with correspondingtabulated peak data shown in Table 5.

TABLE 5 Peak Data of PXRD pattern of crystalline Form 5 Angle, 2θ dspacing Intensity, % 8.04 10.99 100 8.79 10.06 2.2 9.64 9.17 4.2 10.948.08 5 12.15 7.28 7.2 13.09 6.76 10.9 15.07 5.87 9.5 16.04 5.52 6.916.25 5.45 6.9 17.67 5.01 2.2 18.76 4.73 4.4 19.20 4.62 4.8 19.81 4.483.6 21.84 4.07 4.1 23.06 3.85 6 23.87 3.72 11.8 24.10 3.69 17.1 24.493.63 8.8 25.43 3.50 3.8 26.60 3.35 4.5 27.91 3.19 2.6 28.36 3.14 4.929.36 3.04 3.5

The crystalline Form 5 may further be characterized by a DSC thermogramsubstantially the same as that depicted in FIG. 15. As shown in FIG. 15,the crystalline Form 5 has a melting point of about 267° C.; thecrystalline Form 5 has a differential scanning calorimetry thermogramwith endothermic peaks at about 70° C. and at about 267° C.

Plinabulin Crystalline Form 6

Some embodiments relate to a crystalline Form 6 of plinabulin and itsprocess of preparation.

In some embodiments, crystalline Form 6 of plinabulin has substantiallythe same XRPD pattern as depicted in FIG. 16, with correspondingtabulated peak data shown in Table 6.

TABLE 6 Peak Data of PXRD pattern of crystalline Form 6 Angle, 2θ dspacing Intensity, % 6.18 14.28 8.6 8.21 10.77 32.1 8.49 10.41 100 9.569.25 3.8 11.59 7.63 21.4 12.32 7.18 9.9 13.20 6.70 11 13.69 6.46 6.614.90 5.94 57.4 16.08 5.51 8.1 16.68 5.31 5.7 17.20 5.15 27.9 17.33 5.1121 17.68 5.01 17.4 18.19 4.87 10.5 18.50 4.79 16.6 19.53 4.54 18.6 20.844.26 7.1 21.40 4.15 69.3 22.40 3.97 34.7 23.30 3.81 4.9 24.28 3.66 824.76 3.59 6.9 25.25 3.52 6.2 26.57 3.35 3.2 27.05 3.29 5.4 27.68 3.2210.2 28.55 3.12 4.9

The crystalline Form 6 may further be characterized by a DSC thermogramsubstantially the same as that depicted in FIG. 18. As shown in FIG. 18,the crystalline Form 6 has a melting point of about 267° C.; thecrystalline Form 6 has a differential scanning calorimetry thermogramwith endothermic peak at about 267° C.

Plinabulin Crystalline Form 7

Some embodiments relate to a crystalline Form 7 of plinabulin and itsprocess of preparation.

In some embodiments, crystalline Form 7 of plinabulin has substantiallythe same XRPD pattern as depicted in FIG. 19, with correspondingtabulated peak data shown in Table 7.

TABLE 7 Peak Data of PXRD pattern of crystalline Form 7 Angle, 2θ dspacing Intensity, % 6.18 14.28 8.6 8.21 10.77 32.1 8.49 10.41 100 9.569.25 3.8 11.59 7.63 21.4 12.32 7.18 9.9 13.20 6.70 11 13.69 6.46 6.614.90 5.94 57.4 16.08 5.51 8.1 16.68 5.31 5.7 17.20 5.15 27.9 17.33 5.1121 17.68 5.01 17.4 18.19 4.87 10.5 18.50 4.79 16.6 19.53 4.54 18.6 20.844.26 7.1 21.40 4.15 69.3 22.40 3.97 34.7 23.30 3.81 4.9 24.28 3.66 824.76 3.59 6.9 25.25 3.52 6.2 26.57 3.35 3.2 27.05 3.29 5.4 27.68 3.2210.2 28.55 3.12 4.9

The crystalline Form 7 may further be characterized by a DSC thermogramsubstantially the same as that depicted in FIG. 21. As shown in FIG. 21,the crystalline Form 7 has a melting point of about 267° C.; thecrystalline Form 7 has a differential scanning calorimetry thermogramwith endothermic peaks at about 63° C. and at about 267° C.

Plinabulin Crystalline Form 8

Some embodiments relate to a crystalline Form 8 of plinabulin and itsprocess of preparation.

In some embodiments, crystalline Form 8 of plinabulin has substantiallythe same XRPD pattern as depicted in FIG. 22, with correspondingtabulated peak data shown in Table 8.

TABLE 8 Peak Data of PXRD pattern of crystalline Form 8 Angle, 2θ dspacing Intensity, % 6.18 14.28 8.6 8.21 10.77 32.1 8.49 10.41 100 9.569.25 3.8 11.59 7.63 21.4 12.32 7.18 9.9 13.20 6.70 11 13.69 6.46 6.614.90 5.94 57.4 16.08 5.51 8.1 16.68 5.31 5.7 17.20 5.15 27.9 17.33 5.1121 17.68 5.01 17.4 18.19 4.87 10.5 18.50 4.79 16.6 19.53 4.54 18.6 20.844.26 7.1 21.40 4.15 69.3 22.40 3.97 34.7 23.30 3.81 4.9 24.28 3.66 824.76 3.59 6.9 25.25 3.52 6.2 26.57 3.35 3.2 27.05 3.29 5.4 27.68 3.2210.2 28.55 3.12 4.9

The crystalline Form 8 may further be characterized by a DSC thermogramsubstantially the same as that depicted in FIG. 24. As shown in FIG. 24,the crystalline Form 8 has a melting point of about 262° C.; thecrystalline Form 8 has a differential scanning calorimetry thermogramwith endothermic peaks at about 74° C. and at about 264° C.

Plinabulin Crystalline Form 9

Some embodiments relate to a crystalline Form 9 of plinabulin and itsprocess of preparation.

In some embodiments, crystalline Form 9 of plinabulin has substantiallythe same XRPD pattern as depicted in FIG. 25, with correspondingtabulated peak data shown in Table 9.

TABLE 9 Peak Data of PXRD pattern of crystalline Form 9 Angle, 2θ dspacing Intensity, % 7.03 12.57 43 8.00 11.04 100 8.27 10.68 48.3 8.849.99 63.1 9.08 9.74 38.4 9.94 8.89 12.8 11.11 7.96 4.4 12.03 7.35 24.613.32 6.64 5 14.07 6.29 5.1 14.64 6.05 17.4 15.56 5.69 10 16.02 5.5324.2 16.21 5.46 26.6 16.57 5.35 24.4 17.45 5.08 43.7 17.74 4.99 39.418.24 4.86 21.5 18.50 4.79 46 19.78 4.49 41.2 20.63 4.30 17 21.23 4.1896.5 21.80 4.07 8 22.72 3.91 4.1 23.50 3.78 6.5 24.64 3.61 78.6 25.883.44 14.6 26.68 3.34 4.8 27.46 3.25 9.3 27.99 3.19 16 29.45 3.03 3.2

The crystalline Form 9 may further be characterized by a DSC thermogramsubstantially the same as that depicted in FIG. 27. As shown in FIG. 27,the crystalline Form 9 has a melting point of about 267° C.; thecrystalline Form 9 has a differential scanning calorimetry thermogramwith endothermic peaks at about 63° C., about 119° C., about 267° C.,and about 289° C.

Conversion of Plinabulin Polymorph Forms

The plinabulin monohydrate (Form 1) is the most stable polymorph amongthe nine polymorph forms identified. The plinabulin monohydrate (Form 1)remains stable during the drying process and under humidity-basedstability studies (stable to drying at 50° C. under vacuum over theweekend, no change in solid form on exposure to humidity higher than 95%RH for 13 days).

FIG. 28 shows how Form 1 can be converted to the other eight forms ofplinabulin polymorph forms. For example, the plinabulin monohydrate(Form 1) can be converted to Form 2 by slurring Form 1 in isopropylalcohol (about 10 times the volume of Form 1) at 30° C. for 3 hours;Form 1 can be converted to Form 3 by slurring Form 1 in ethanol (about10 times the volume of Form 1) at room temperature for overnight; Form 1can be converted to Form 4 by slurring Form 1 in methanol (about 10times the volume of Form 1) at 30° C. for overnight; Form 1 can beconverted to Form 5 by slurring Form 1 in acetonitrile (ACN) at 30° C.and stirring for 3 days; Form 1 can be converted to Form 6 by preparinga Form 1 saturated isopropyl alcohol solution at 15° C. and thenevaporate in vacuum over at 45° C.; Form 1 can be converted to Form 7 bypreparing a Form 1 saturated methanol solution at 45° C. and thenevaporate in vacuum over at 45° C.; Form 1 can be converted to Form 8 bypreparing a Form 1 saturated ethyl acetate (EtOAc) solution at 45° C.and then evaporate in vacuum over at 45° C.; and Form 1 can be convertedto Form 9 by first converting Form 1 to Form 4 and then exposing Form 4to moisture.

FIG. 28 also shows how other forms can be converted to Form 1. Forexample, Forms 2, 3, and 4 can be converted to Form 1 by slurrying theseforms in a mixture of ethanol and water (95:5 by volume) (the volume ofethanol and water mixture is about 10 times of the staring polymorphforms) at 30° C. for 2 hours; Form 2 can be converted to Form 6 bydrying it in vacuum oven at 45° C. for 5 days; Form 4 can be convertedto Form 7 by drying it in vacuum oven at 45° C. overnight; and Form 4can be converted to Form 9 by exposing it to high humidity.

Administration and Pharmaceutical Compositions

Some embodiments include pharmaceutical compositions comprising theplinabulin polymorph described herein and a pharmaceutically acceptablecarrier. Such a composition can be administered to a subject as part ofa therapeutic treatment.

In some embodiments, the composition can further include one or morepharmaceutically acceptable diluents. In some embodiments, thepharmaceutically acceptable diluent can include Kolliphor® (Polyoxyl 15hydroxystearate). In some embodiments, the pharmaceutically acceptablediluent can include propylene glycol. In some embodiments, thepharmaceutically acceptable diluents can include kolliphor and propyleneglycol. In some embodiments, the pharmaceutically acceptable diluentscan include kolliphor and propylene glycol, wherein the kolliphor isabout 40% by weight and propylene glycol is about 60% by weight based onthe total weight of the diluents. In some embodiments, the compositioncan further include one or more other pharmaceutically acceptableexcipients.

Standard pharmaceutical formulation techniques can be used to make thepharmaceutical compositions described herein, such as those disclosed inRemington's The Science and Practice of Pharmacy, 21st Ed., LippincottWilliams & Wilkins (2005), incorporated herein by reference in itsentirety. Accordingly, some embodiments include pharmaceuticalcompositions comprising: (a) a safe and therapeutically effective amountof Plinabulin polymorph or pharmaceutically acceptable salts thereof;and (b) a pharmaceutically acceptable carrier, diluent, excipient orcombination thereof.

Other embodiments include co-administering plinabulin polymorph and anadditional therapeutic agent in separate compositions or the samecomposition. Thus, some embodiments include a first pharmaceuticalcompositions comprising: (a) a safe and therapeutically effective amountof plinabulin polymorph or pharmaceutically acceptable salts thereof and(b) a pharmaceutically acceptable carrier, diluent, excipient orcombination thereof; and a second pharmaceutical composition comprising:(a) a safe and therapeutically effective amount of an additionaltherapeutic agent and (b) a pharmaceutically acceptable carrier,diluent, excipient or combination thereof. Some embodiments include apharmaceutical composition comprising: (a) a safe and therapeuticallyeffective amount of plinabulin polymorph or pharmaceutically acceptablesalts thereof; (b) a safe and therapeutically effective amount of anadditional therapeutic agent; and (c) a pharmaceutically acceptablecarrier, diluent, excipient or combination thereof.

Administration of the pharmaceutical compositions described herein canbe via any of the accepted modes of administration for agents that servesimilar utilities including, but not limited to, orally, sublingually,buccally, subcutaneously, intravenously, intranasally, topically,transdermally, intradermally, intraperitoneally, intramuscularly,intrapulmonarilly, vaginally, rectally, or intraocularly. Oral andparenteral administrations are customary in treating the indicationsthat are the subject of the preferred embodiments.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. In addition, various adjuvants such as are commonly usedin the art may be included. Considerations for the inclusion of variouscomponents in pharmaceutical compositions are described, e.g., in Gilmanet al. (Eds.) (1990); Goodman and Gilman's: The Pharmacological Basis ofTherapeutics, 8th Ed., Pergamon Press, which is incorporated herein byreference in its entirety.

Some examples of substances, which can serve aspharmaceutically-acceptable carriers or components thereof, are sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powderedtragacanth; malt; gelatin; talc; solid lubricants, such as stearic acidand magnesium stearate; calcium sulfate; vegetable oils, such as peanutoil, cottonseed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerine, sorbitol,mannitol, and polyethylene glycol; alginic acid; emulsifiers, such asthe TWEENS; wetting agents, such sodium lauryl sulfate; coloring agents;flavoring agents; tableting agents, stabilizers; antioxidants;preservatives; pyrogen-free water; isotonic saline; and phosphate buffersolutions.

The compositions described herein are preferably provided in unit dosageform. As used herein, a “unit dosage form” is a composition containingan amount of a compound or composition that is suitable foradministration to an animal, preferably a mammalian subject, in a singledose, according to good medical practice. The preparation of a single orunit dosage form however, does not imply that the dosage form isadministered once per day or once per course of therapy. Such dosageforms are contemplated to be administered once, twice, thrice or moreper day and may be administered as infusion over a period of time (e.g.,from about 30 minutes to about 2-6 hours), or administered as acontinuous infusion, and may be given more than once during a course oftherapy, although a single administration is not specifically excluded.The skilled artisan will recognize that the formulation does notspecifically contemplate the entire course of therapy and such decisionsare left for those skilled in the art of treatment rather thanformulation.

The compositions useful as described above may be in any of a variety ofsuitable forms for a variety of routes for administration, for example,for oral, sublingual, buccal, nasal, rectal, topical (includingtransdermal and intradermal), ocular, intracerebral, intracranial,intrathecal, intra-arterial, intravenous, intramuscular, or otherparental routes of administration. The skilled artisan will appreciatethat oral and nasal compositions include compositions that areadministered by inhalation, and made using available methodologies.Depending upon the particular route of administration desired, a varietyof pharmaceutically-acceptable carriers well-known in the art may beused. Pharmaceutically-acceptable carriers include, for example, solidor liquid fillers, diluents, hydrotropies, surface-active agents, andencapsulating substances. Optional pharmaceutically-active materials maybe included, which do not substantially interfere with the activity ofthe compound or composition. The amount of carrier employed inconjunction with the compound or composition is sufficient to provide apractical quantity of material for administration per unit dose of thecompound. Techniques and compositions for making dosage forms useful inthe methods described herein are described in the following references,all incorporated by reference herein: Modern Pharmaceutics, 4th Ed.,Chapters 9 and 10 (Banker & Rhodes, editors, 2002); Lieberman et al.,Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction toPharmaceutical Dosage Forms 8th Edition (2004).

Various oral dosage forms can be used, including such solid forms astablets, capsules (e.g., liquid gel capsule and solid gel capsule),granules and bulk powders. Tablets can be compressed, tablet triturates,enteric-coated, sugar-coated, film-coated, or multiple-compressed,containing suitable binders, lubricants, diluents, disintegratingagents, coloring agents, flavoring agents, flow-inducing agents, andmelting agents. Liquid oral dosage forms include aqueous solutions,emulsions, suspensions, solutions and/or suspensions reconstituted fromnon-effervescent granules, and effervescent preparations reconstitutedfrom effervescent granules, containing suitable solvents, preservatives,emulsifying agents, suspending agents, diluents, sweeteners, meltingagents, coloring agents and flavoring agents.

The pharmaceutically-acceptable carriers suitable for the preparation ofunit dosage forms for peroral administration is well-known in the art.Tablets typically comprise conventional pharmaceutically-compatibleadjuvants as inert diluents, such as calcium carbonate, sodiumcarbonate, mannitol, lactose and cellulose; binders such as starch,gelatin and sucrose; disintegrants such as starch, alginic acid andcroscarmelose; lubricants such as magnesium stearate, stearic acid andtalc. Glidants such as silicon dioxide can be used to improve flowcharacteristics of the powder mixture. Coloring agents, such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, sucrose, and fruitflavors, are useful adjuvants for chewable tablets. Capsules typicallycomprise one or more solid diluents disclosed above. The selection ofcarrier components depends on secondary considerations like taste, cost,and shelf stability, which are not critical, and can be readily made bya person skilled in the art.

Peroral compositions also include liquid solutions, emulsions,suspensions, and the like. The pharmaceutically-acceptable carrierssuitable for preparation of such compositions are well known in the art.Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, AVICEL RC-591, tragacanth and sodium alginate; typicalwetting agents include lecithin and polysorbate 80; and typicalpreservatives include methyl paraben and sodium benzoate. Peroral liquidcompositions may also contain one or more components such as sweeteners,flavoring agents and colorants disclosed above.

Such compositions may also be coated by conventional methods, typicallywith pH or time-dependent coatings, such that the subject composition isreleased in the gastrointestinal tract in the vicinity of the desiredtopical application, or at various times to extend the desired action.Such dosage forms typically include, but are not limited to, one or moreof cellulose acetate phthalate, polyvinylacetate phthalate,hydroxypropyl methyl cellulose phthalate, ethyl cellulose, Eudragitcoatings, waxes and shellac.

Compositions described herein may optionally include other drug actives.

Other compositions useful for attaining systemic delivery of the subjectcompounds include sublingual, buccal and nasal dosage forms. Suchcompositions typically comprise one or more of soluble filler substancessuch as sucrose, sorbitol and mannitol; and binders such as acacia,microcrystalline cellulose, carboxymethyl cellulose and hydroxypropylmethyl cellulose. Glidants, lubricants, sweeteners, colorants,antioxidants and flavoring agents disclosed above may also be included.

A liquid composition, which is formulated for topical ophthalmic use, isformulated such that it can be administered topically to the eye. Thecomfort may be maximized as much as possible, although sometimesformulation considerations (e.g. drug stability) may necessitate lessthan optimal comfort. In the case that comfort cannot be maximized, theliquid may be formulated such that the liquid is tolerable to thepatient for topical ophthalmic use. Additionally, an ophthalmicallyacceptable liquid may either be packaged for single use, or contain apreservative to prevent contamination over multiple uses.

For ophthalmic application, solutions or medicaments are often preparedusing a physiological saline solution as a major vehicle. Ophthalmicsolutions may preferably be maintained at a comfortable pH with anappropriate buffer system. The formulations may also containconventional, pharmaceutically acceptable preservatives, stabilizers andsurfactants.

Preservatives that may be used in the pharmaceutical compositionsdisclosed herein include, but are not limited to, benzalkonium chloride,PHMB, chlorobutanol, thimerosal, phenylmercuric, acetate andphenylmercuric nitrate. A useful surfactant is, for example, Tween 80.Likewise, various useful vehicles may be used in the ophthalmicpreparations disclosed herein. These vehicles include, but are notlimited to, polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose,poloxamers, carboxymethyl cellulose, hydroxyethyl cellulose and purifiedwater.

Tonicity adjustors may be added as needed or convenient. They include,but are not limited to, salts, particularly sodium chloride, potassiumchloride, mannitol and glycerin, or any other suitable ophthalmicallyacceptable tonicity adjustor.

Various buffers and means for adjusting pH may be used so long as theresulting preparation is ophthalmically acceptable. For manycompositions, the pH will be between 4 and 9. Accordingly, buffersinclude acetate buffers, citrate buffers, phosphate buffers and boratebuffers. Acids or bases may be used to adjust the pH of theseformulations as needed.

Ophthalmically acceptable antioxidants include, but are not limited to,sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole and butylated hydroxytoluene.

Other excipient components, which may be included in the ophthalmicpreparations, are chelating agents. A useful chelating agent is edetatedisodium (EDTA), although other chelating agents may also be used inplace or in conjunction with it.

For topical use, creams, ointments, gels, solutions or suspensions,etc., containing the composition disclosed herein are employed. Topicalformulations may generally be comprised of a pharmaceutical carrier,co-solvent, emulsifier, penetration enhancer, preservative system, andemollient.

For intravenous administration, the compositions described herein may bedissolved or dispersed in a pharmaceutically acceptable diluent, such asa saline or dextrose solution. Suitable excipients may be included toachieve the desired pH, including but not limited to NaOH, sodiumcarbonate, sodium acetate, HCl, and citric acid. In various embodiments,the pH of the final composition ranges from 2 to 8, or preferably from 4to 7. Antioxidant excipients may include sodium bisulfite, acetonesodium bisulfite, sodium formaldehyde, sulfoxylate, thiourea, and EDTA.Other non-limiting examples of suitable excipients found in the finalintravenous composition may include sodium or potassium phosphates,citric acid, tartaric acid, gelatin, and carbohydrates such as dextrose,mannitol, and dextran. Further acceptable excipients are described inPowell, et al., Compendium of Excipients for Parenteral Formulations,PDA J Pharm Sci and Tech 1998, 52 238-311 and Nema et al., Excipientsand Their Role in Approved Injectable Products: Current Usage and FutureDirections, PDA J Pharm Sci and Tech 2011, 65 287-332, both of which areincorporated herein by reference in their entirety. Antimicrobial agentsmay also be included to achieve a bacteriostatic or fungistaticsolution, including but not limited to phenylmercuric nitrate,thimerosal, benzethonium chloride, benzalkonium chloride, phenol,cresol, and chlorobutanol.

The compositions for intravenous administration may be provided tocaregivers in the form of one more solids that are reconstituted with asuitable diluent such as sterile water, saline or dextrose in watershortly prior to administration. In other embodiments, the compositionsare provided in solution ready to administer parenterally. In stillother embodiments, the compositions are provided in a solution that isfurther diluted prior to administration. In embodiments that includeadministering a combination of a compound described herein and anotheragent, the combination may be provided to caregivers as a mixture, orthe caregivers may mix the two agents prior to administration, or thetwo agents may be administered separately.

In some embodiments, a single dose of plinabulin polymorph or othertherapeutic agent may be from about 5 mg/m² to about 150 mg/m² of bodysurface area, from about 5 mg/m² to about 100 mg/m² of body surfacearea, from about 10 mg/m² to about 100 mg/m² of body surface area, fromabout 10 mg/m² to about 80 mg/m² of body surface area, from about 10mg/m² to about 50 mg/m² of body surface area, from about 10 mg/m² toabout 40 mg/m² of body surface area, from about 10 mg/m² to about 30mg/m² of body surface area, from about 13.5 mg/m² to about 100 mg/m² ofbody surface area, from about 13.5 mg/m² to about 80 mg/m² of bodysurface area, from about 13.5 mg/m² to about 50 mg/m² of body surfacearea, from about 13.5 mg/m² to about 40 mg/m² of body surface area, fromabout 13.5 mg/m² to about 30 mg/m² of body surface area, from about 15mg/m² to about 80 mg/m² of body surface area, from about 15 mg/m² toabout 50 mg/m² of body surface area, or from about 15 mg/m² to about 30mg/m² of body surface area. In some embodiments, a single dose ofplinabulin polymorph or other therapeutic agent may be from about 13.5mg/m² to about 30 mg/m² of body surface area. In some embodiments, asingle dose of plinabulin polymorph or other therapeutic agent may beabout 5 mg/m², about 10 mg/m², about 12.5 mg/m², about 13.5 mg/m², about15 mg/m², about 17.5 mg/m², about 20 mg/m², about 22.5 mg/m², about 25mg/m², about 27.5 mg/m², about 30 mg/m², about 40 mg/m², about 50 mg/m²,about 60 mg/m², about 70 mg/m², about 80 mg/m², about 90 mg/m², or about100 mg/m², of body surface area.

In some embodiments, a single dose of plinabulin polymorph or othertherapeutic agent may be from about 5 mg to about 300 mg, from about 5mg to about 200 mg, from about 7.5 mg to about 200 mg, from about 10 mgto about 100 mg, from about 15 mg to about 100 mg, from about 20 mg toabout 100 mg, from about 30 mg to about 100 mg, from about 40 mg toabout 100 mg, from about 10 mg to about 80 mg, from about 15 mg to about80 mg, from about 20 mg to about 80 mg, from about 30 mg to about 80 mg,from about 40 mg to about 80 mg, from about 10 mg to about 60 mg, fromabout 15 mg to about 60 mg, from about 20 mg to about 60 mg, from about30 mg to about 60 mg, or from about 40 mg to about 60 mg, In someembodiments, a single dose of plinabulin polymorph or other therapeuticagent may be from about 20 mg to about 60 mg, from about 27 mg to about60 mg, from about 20 mg to about 45 mg, or from about 27 mg to about 45mg. In some embodiments, a single dose of plinabulin polymorph or othertherapeutic agent may be about 5 mg, about 10 mg, about 12.5 mg, about13.5 mg, about 15 mg, about 17.5 mg, about 20 mg, about 22.5 mg, about25 mg, about 27 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg,about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about150 mg, or about 200 mg.

The administration period can be a multi-week treatment cycle as long asthe tumor remains under control and the regimen is clinically tolerated.In some embodiments, a single dosage of plinabulin polymorph or othertherapeutic agent can be administered once a week, and preferably onceon each of day 1 and day 8 of a three-week (21 day) treatment cycle. Insome embodiments, a single dosage of plinabulin polymorph or othertherapeutic agent can be administered once a week, twice a week, threetimes per week, four times per week, five times per week, six times perweek, or daily during a one-week, two-week, three-week, four-week, orfive-week treatment cycle. The administration can be on the same ordifferent day of each week in the treatment cycle.

The treatment cycle can be repeated as long as the regimen is clinicallytolerated. In some embodiments, the treatment cycle is repeated for ntimes, wherein n is an integer in the range of 2 to 30. In someembodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, anew treatment cycle can occur immediately after the completion of theprevious treatment cycle. In some embodiments, a new treatment cycle canoccur a period of time after the completion of the previous treatmentcycle.

In some embodiments, the compositions described herein can be used incombination with other therapeutic agents. In some embodiments, thecompositions described herein can be administered or used in combinationwith treatments such as chemotherapy, radiation, and biologic therapies.

EXAMPLES Example 1

A sample of plinabulin compound was stirred in ethanol and heated toreflux. Ethanol was added in portions to maintain reflux until theentire sampled dissolved to give a clear yellow solution. A total of124.7 g of ethanol were required to completely dissolve the sample atreflux. The solution was then allowed to cool and monitored forprecipitation. A precipitate was observed when the solution was at 49°C. The mixture was reheated to reflux providing a clear yellow solution.The hot solution was transferred to a larger Erlenmeyer flask with a9.45 g ethanol rinse (to mimic a hot filtration). To this refluxingsolution was added 6.6 g of water (approximately 5% water in ethanol).This solution was allowed to cool slowly with stirring. A precipitatewas observed when the solution cooled to 70° C. At this point additionalwater (128.6 g) was added slowly causing a large amount of solid toprecipitate. The solution was allowed to cool to room temperature withstirring. The solids were filtered at 17° C. and washed three times with20 g of water. A total of 134 g of water was added to the filtratecausing a hazy solution, an insufficient amount of solid to filter. Anadditional 137 g of water was added in an attempt to precipitateadditional product but no additional solid was recoverable. The solidwas dried at 40-45° C. for 3 days to give 4.73 g, 97.5% recovery.Analysis by XRPD showed the product to be plinabulin monohydrate (Form1). Analysis by Karl Fischer showed the moisture level was 4.9%.

Analysis by KF after drying at 45° C. under vacuum for an additional 70hours indicated the moisture level had decreased to 4.1%. The XRPD ofthis sample indicated that it was plinabulin monohydrate (Form 1). Thissample was placed in a glove bag in an open container with an opencontainer of water and the moisture level monitored. After 4 hours themoisture level was measured as 5.0%. After 18.5 hours the moisture levelwas measured as 4.8% and after 51 hours the KF result was 4.9%.

Example 2

A sample of plinabulin compound (4.92 g) was stirred in ethanol (147.6g) and heated to reflux (fully soluble at 75° C.). The solution was thenallowed to cool and monitored for precipitation. A precipitate wasobserved when the solution was at 48° C. The mixture was re-heated toreflux providing a clear yellow solution. To the hot solution was added295 g of water (approximately twice the mass of ethanol) allowing themixture to cool during the addition. A precipitate was observed aftercharging approximately 150 mL of water with the temperature at 48° C.This solution was allowed to cool to room temperature. The solids werefiltered and washed three times with 20 g of water. The solid was driedat 40-45° C. for 2.5 days to give 4.82 g, 98.0% recovery. Analysis byXRPD showed the product to be a mix of plinabulin monohydrate (Forms 1)(major) and anhydrous plinabulin (Form 3) (minor). Analysis by KarlFischer showed the moisture level was 5.0%. Analysis by KF after dryingat 45° C. under vacuum for an additional 70 hours indicated the moisturelevel had decreased to 4.4%. The XRPD of this sample indicates it wasessentially unchanged, a mixture of Forms 1 and 3 with additional peaks(at around 12.26°, 15.19° and 28.79° 2θ degrees). This sample was placedin a glove bag in an open container with an open container of water andthe moisture level monitored. After 4 hours the moisture level wasmeasured as 5.0%. After 18.5 hours the moisture level was measured as4.9% and after 51 hours the KF result was 5.1%.

Example 3

A sample of plinabulin compound was dissolved in 1,2-propanediol withvigorous stirring or mild heating (50° C.). After 4 hours, the sampleshowed peaks associated with Form 3. After stirring over the weekend,the sample was slurried in water and converted entirely to plinabulinmonohydrate (Form 1) with no peaks associated with crystalline Form 3present in the XRPD scans. A similar result was seen for the experimentin ethanol/water, with very small peaks seen for Form 3 at 1 hour and 4hours and only Form 1 observed after 66 hours.

Example 4

In a reprocessing procedure, a plinabulin compound (Form 3) wasdissolved in ethanol at a ratio of 1:25 (by weight) at reflux. Thissolution was filtered at a temperature that was higher 50° C. and thefiltrate was combined with an equal mass of water to afford the product.It may be desirable to reheat the polish-filtered ethanol solution priorto addition of the entire amount of water and add approximately 5% water(relative to ethanol) and stir this solution at approximately 70° C. toensure conversion to Form 1. Additional water can then be added and themixture cooled to isolate the product by filtration. The sample can bedried for an extended period to lower the moisture content as measuredby Karl Fisher analysis. One sample was dried for three days resultingin a KF analysis of 4.9% moisture. Drying for an additional three dayslowered the KF result to 4.1%.

Exposing the dry product to a humid environment raised the moisturelevel to approximately 5% where it appeared to remain stable. One samplewith 4.1% moisture was exposed to an open container of water in a glovebag for 4 hours, raising the moisture level to 5.0%. After an additional14.5 hours in this environment the moisture level in the sample wasmeasured as 4.8%. After a total of 51 hours the KF result was 4.9%.

Example 5

A batch of plinabulin having a KF analysis of about 3.1% for watercontent was added to a mixture of kolliphor (40% wt) and propyleneglycol (60% wt). Insoluble particles were formed in the solution, and itwas determined that the insoluble particles were anhydrous plinabulin(Form 3). The batch was reprocessed according to the steps described inFIG. 29 to form the plinabulin monohydrate (Form 1). Analysis by KFshowed that the water content of the reprocessed plinabulin was about5.1%, which is consistent with the theoretical water content for themonohydrate. The plinabulin monohydrate (Form 1) dissolved completely ina mixture of kolliphor (40% wt) and propylene glycol (60% wt) and noinsoluble particles were formed in the solution. Therefore, theplinabulin monohydrate (Form 1) showed better solubility than plinabulincompositions that contain anhydrous plinabulin (Form 3).

Example 6

The plinabulin monohydrate (Form 1 crystalline) was characterized byXRPD (crystalline, FIG. 1), optical microscopy (FIG. 2), DSC (FIG. 3A),TGA (FIG. 3B), and KF.

The DSC data were collected using a TA Instruments Q10 DSC. Typically,samples (˜2 mg) were placed in hermetic alodined aluminum sample pansand scanned from 30 to 300° C. at a rate of 10° C./min under a nitrogenpurge of 50 mL/min. The TGA data were collected using a TA InstrumentsTGA Q500. Typically, samples (˜10 mg) were placed in an open, pre-taredaluminum sample pan and scanned from 30 to 300° C. at a rate of 10°C./min using a nitrogen purge at 60 mL/min. The X-ray powder diffractionpatterns were obtained using a Bruker D8 Advance equipped with a Cu Kαradiation source (1.54° A), a 9-position sample holder and a LYNXEYESuper Speed Detector. Typically, the duration of each scan was 180seconds and the 20 range was 4 to 40°. Samples were placed onzero-background, silicon plate holders. Samples were analyzed using anAquadyne DVS-2 gravimetric water sorption analyzer. The relativehumidity was adjusted between 2-95% and the weight of sample wascontinuously monitored and recorded.

The XRPD showed that the material is crystalline. The DSC data showed abroad endotherm (peak max at 141° C., likely water loss), a smallexothermic event (peak max 164° C.) and a sharp endothermic event (peakmax 268° C.). TGA indicated loss of 5.26% of mass at about 130° C.(likely water loss). KF analysis also showed that the material containswater at 5.25 weight %.

A sample of plinabulin monohydrate (Form 1) was placed in a vacuum ovenat 50° C. overnight and over the weekend. The sample remained stableduring the drying studies and no change in weight occurred during thedrying process.

A DVS experiment was run on the plinabulin monohydrate (Form 1). Thesample gained ˜0.1% mass at 95% RH which was lost on drying to 0% RHwithout hysteresis. The post-DVS sample was analyzed by XRPD whichconfirmed that no transformation had taken place. FIG. 3C shows the DVSisotherm plot. The XRPD pattern of the pre-DVS sample (Form 1 plinabulinmonohydrate) overlays with the XRPD patterns of the post-DVS sample(Form 1 plinabulin monohydrate).

The DVS test of Form 2 solids showed that the Form 2 sample lost about7% mass at about 80% RH, and the post-DVS sample differed from thepre-DVS sample based on the XRPD pattern analysis.

The DVS test of Form 3 solids showed that the Form 3 sample lost about0.2% mass at 90% RH, and the post-DVS sample differed from the pre-DVSsample based on the XRPD pattern analysis.

The DVS test of Form 4 solids showed that the Form 3 sample lost mass inthe test. The post-DVS sample differed from the pre-DVS sample based onthe XRPD pattern analysis.

The plinabulin monohydrate (Form 1) remained stable during the DVS anddrying tests. In comparison, other polymorph forms were unstable andshowed weight change during the DVS tests. The test results have shownthat Form 1 is more stable than the other polymorph forms.

Example 7

A plinabulin compound was slurried in 15 different solvents/solventmixtures as shown in Table 9 at 15° C. and 45° C. for 3 days forgravimetric solubility measurement. About 70 mg of solid was added to avial for each experiment followed by addition of 0.7 mL of therespective solvents. Next, the slurry was centrifuged and thesupernatant was added to pre-weighed vials and evaporated to drynessunder vacuum. The vials with remaining solids were weighed again tocalculate the solubility. The compound is highly soluble in THF and hadmoderate to low solubility in the other solvents tested. The solubilitydata is shown in Table 10.

TABLE 10 Gravimetric solubility data of plinabulin monohydrate invarious solvents. Solubility at Solubility at Sample No. Solvent 15° C.in mg/mL 45° C. in mg/mL 1 Heptane <1 3 2 Toluene 1 7 3 MTBE <1 5 4EtOAc 3 8 5 THF 40 96 6 IPA 1 7 7 Acetone 11 20 8 EtOH 5 13 9 MeOH 5 1310 ACN 3 7 11 Water <1 <1 12 MEK 11 23 13 DCM 2 N/A 14 Acetone:water 616 (95:5) 15 EtOH:water (95:5) 4 13

During gravimetric solubility analysis, the plinabulin monohydrate(Form 1) was slurried in various solvents at 15° C. and at 45° C. Aftercentrifugation, the solids obtained were analyzed as wet cake by XRPD.The samples which were not Form 1 were dried in a vacuum oven andreanalyzed by XRPD. Forms 2, 3, 4, 5, 6, and 7 were observed. Theseresults are shown in Table 11.

TABLE 11 XRPD results of slurry experiments in various solvents beforeand after drying 15° C. slurry 45° C. slurry 15° C. slurry 45° C. slurryNo. Solvent wet XRPD wet XRPD dry XRPD dry XRPD 1 Heptane Form 1 Form 1N/A N/A 2 Toluene Form 1 Form 1 N/A N/A 3 MTBE Form 1 Form 1 N/A N/A 4EtOAc Form 1 Form 1 + 3 N/A Still Form 1 + 3 5 THF Form 1 Form 1 + 3 N/AStill Form 1 + 3 6 IPA Form 2 Form 1 + 2 Form 6 Form 6 + 2 7 AcetoneForm 1 + 3 Form 3 Still Form 1 + 3 Still Form 3 8 EtOH Form 3 Form 3Still Form 3 Still Form 3 9 MeOH Form 1 + 4 Form 4 Still Form 1 + 4 Form7 10 ACN Form 5 Form 3 Still Form 5 N/A 11 Water Form 1 Form 1 N/A N/A12 MEK Form 1 Form 3 N/A N/A 13 DCM Form 1 N/A N/A N/A 14 Acetone:waterForm 1 Form 1 N/A N/A (95:5) 15 EtOH:water Form 1 Form 1 N/A N/A (95:5)

Evaporation crystallization experiments were setup by evaporating (atroom temperature) solutions of the plinabulin monohydrate in varioussolvents. These solutions were obtained during the gravimetricsolubility analysis at 15° C. and at 45° C. and were of differentconcentrations. The solids obtained were analyzed by XRPD. Form 8 wasobserved. These results are shown in Table 12.

TABLE 12 XRPD results of evaporation crystallization experimentsEvaporation of Evaporation of 15° C. solubility 45° C. solubility No.Solvent samples samples 1 Heptane Not enough solids Not enough solids 2Toluene Not enough solids Not enough solids 3 MTBE Not enough solids Notenough solids 4 EtOAc Not enough solids Form 8 5 THF Form 6 Form 8 6 IPANot enough solids Form 6 7 Acetone Form 3 + 6 Form 8 8 EtOH Not enoughsolids Form 3 9 MeOH Mostly amorphous Form 7 w/Form 8 peak 10 ACN Notenough solids Form 3 11 Water Not enough solids Not enough solids 12 MEKForm 3 + 6 Form 3 13 DCM Not enough solids N/A 14 Acetone:water Form 8Form 8 (95:5) 15 EtOH:water (95:5) Low Crystallinity Form 6 Form 1

Six cooling crystallization experiments and one slurry experiment werecarried out. The results of these experiments are shown in Table 13.

TABLE 13 XRPD results of cooling crystallization experiments SolventProcedure XRPD 1 THF 100 mg solids dissolved in 10 vol. Form 1 + 5solvent at 60° C. Cooled naturally to RT and stirred for 2 hours. Cooledto 5° C. using a chiller and stirred for 3 hours. Crystallized. 2THF:acetone 1:1 100 mg solids dissolved in 16 vol. Form 1 solvent at 60°C. Cooled naturally to RT and stirred for 2 hours. Cooled to 5° C. usinga chiller and stirred overnight. Crystallized. 3 THF:EtOH 1:1 100 mgsolids dissolved in 11 vol. Form 3 solvent at 60° C. Cooled naturally toRT and stirred for 2 hours. Cooled to 5° C. using a chiller and stirredfor 2 hours. Crystallized. 4 THF:MeOH 1:1 100 mg solids dissolved in 8vol. Form 4 solvent at 60° C. Cooled naturally to RT and stirred for 2hours. Cooled to 5° C. using a chiller. Crystallized at 12° C. 5THF:water 1:1 100 mg solids did not dissolve in 20 Form 1 vol. solventat 60° C. Cooled naturally to RT and stirred overnight. 6 THF:MEK(methyl 100 mg solids dissolved in 20 vol. Form 1 ethyl ketone) 1:1solvent at 60° C. Cooled naturally to RT and stirred for 2 hours. Cooledto 5° C. using a chiller and stirred for 2 hours. Crystallized.

Example 8

The various crystalline forms were tested for conversion to other formsat a 200 mg scale.

A Form 3 scale up experiment was carried out at the 200 mg scale byslurrying Form 1 solids in 2 mL ethanol at room temperature overnight toproduce Form 3.

A Form 2 scale up experiment was carried out at the 200 mg scale byslurrying Form 1 solids in 2 mL isopropyl alcohol at 30° C. overnight.

A Form 4 scale up experiment was carried out at the 200 mg scale byslurrying Form 1 solids in 2 mL MeOH at 30° C. overnight.

Form 3 solids were slurried in a 95:5 EtOH:water mixture at 30° C. for 2hours and analyzed by XRPD to show conversion to Form 1.

Form 2 solids were slurried (2071-16-1) in a 95:5 EtOH:water mixture at30° C. for 2 hours and analyzed by XRPD to show conversion to Form 1.

Form 4 solids were slurried in a 95:5 EtOH:water mixture at 30° C. for 2hours and analyzed by XRPD to show conversion to Form 1.

The plinabulin monohydrate (Form 1) remained stable when dried at 50° C.under vacuum over the weekend. There was no change in solid form whenform 1 was exposed to high humidity (>95% RH) for 13 days. Form 1 wasshown to be stable during the manufacturing process including the dryingprocess. Form 1 was also stable under various humidity conditions. Onthe other hand, the crystalline forms converted to Form I monohydratewhen exposed to moisture. Therefore, Form 1 is the most stablecrystalline form and the most viable form for manufacturing process.

What is claimed is:
 1. A sterile container, comprising a plinabulinmonohydrate in crystalline solid form, wherein the plinabulinmonohydrate exhibits an X-ray powder diffraction pattern comprising atleast three characteristic peaks selected from the group consisting ofpeaks at approximately 8.1°, 13.1°, 16.3°, 23.9°, 24.2°, 24.5°, and26.6° 2θ.
 2. A process of preparing a plinabulin monohydrate incrystalline form comprising the steps of: combining plinabulin and afirst solvent system to form a first mixture, wherein the first solventsystem is water, alcohol, or a mixture of water and alcohol, and whereinthe alcohol, if present, is selected from the group consisting ofmethanol, ethanol, isopropyl alcohol, tert-butyl alcohol, n-butylalcohol, and any mixtures thereof; heating the first mixture to atemperature in the range of about 50° C. to 90° C.; and cooling thefirst mixture to form a first precipitate, wherein the plinabulinmonohydrate exhibits an X-ray powder diffraction pattern comprising atleast three characteristic peaks selected from the group consisting ofpeaks at approximately 8.1°, 13.1°, 16.3°, 23.9°, 24.2°, 24.5°, and26.6° 2θ.
 3. The process of claim 2, the first solvent system comprisesethanol.
 4. The process of claim 2, wherein the heating step comprises:refluxing the first mixture; or heating the first mixture to atemperature in the range of about 70° C. to 78° C.
 5. The process ofclaim 2, further comprising, prior to the cooling step, maintaining thefirst mixture at a temperature in the range of about 70° C. to 78° C.for about 1 hour.
 6. The process of claim 2, wherein the cooling stepcomprises at least one of: adding water to the first mixture to producethe first precipitate; and stirring the first mixture for at least 4hours.
 7. The process of claim 2, wherein the heating step comprisesheating the first mixture to a temperature of at least 65° C., andwherein the cooling step comprises cooling the first mixture to atemperature in the range of about 50° C. to 60° C.
 8. The process ofclaim 2, further comprising one or more steps each independentlyselected from the group consisting of: filtering the heated firstmixture prior to the first cooling step; filtering the firstprecipitate; washing the first precipitate; and analyzing the firstprecipitate using X-ray powder diffraction analysis.
 9. The process ofclaim 2, further comprising the additional steps of: combining the firstprecipitate and a second solvent to form a second mixture, wherein thesecond solvent is water, alcohol, or a mixture of water and alcohol, andwherein the alcohol, if present, is selected from the group consistingof methanol, ethanol, isopropyl alcohol, tert-butyl alcohol, n-butylalcohol, and mixtures thereof; heating the second mixture to atemperature in the range of about 50° C. to 90° C.; cooling the secondmixture to a temperature in the range of about 15° C. to 30° C. to forma second precipitate; filtering the second precipitate; and washing thesecond precipitate.
 10. The process of claim 9, wherein the secondsolvent is ethanol.
 11. The process of claim 9, wherein the furtherheating step comprises: refluxing the second mixture; or heating thesecond mixture to a temperature in the range of about 70° C. to 78° C.12. The process of claim 9, further comprising, prior to the furthercooling step, maintaining the second mixture at a refluxing temperaturefor about 1 hour.
 13. The process of claim 9, wherein the furthercooling step comprises at least one of: adding water to the secondmixture to produce the second precipitate; and stirring the secondmixture for at least 4 hours.
 14. The process of claim 9, furthercomprising the following steps: washing the first precipitate with analcohol, selected from the group consisting of methanol, ethanol,isopropyl alcohol, tert-butyl alcohol, n-butyl alcohol, and mixturesthereof; collecting the alcohol wash; and adding the collected alcoholwash to the second mixture prior to the further heating step.
 15. Theprocess of claim 9, further comprising one or more steps eachindependently selected from the group consisting of: drying the secondprecipitate; and analyzing the second precipitate using X-ray powderdiffraction analysis.
 16. The process of claim 9, wherein the additionalcombining step, the additional cooling step, and the filtering step arerepeated one or more times based on the amount of plinabulin in thesecond precipitate.
 17. A process of preparing a plinabulin monohydratein crystalline form, comprising mixing plinabulin, ethanol and water toform a mixture, wherein the plinabulin monohydrate exhibits an X-raypowder diffraction pattern comprising at least three characteristicpeaks selected from the group consisting of peaks at approximately 8.1°,13.1°, 16.3°, 23.9°, 24.2°, 24.5°, and 26.6° 2θ.
 18. The process ofclaim 17, wherein the volume ratio of ethanol to water is in the rangeof about 15:1 to about 25:1.
 19. The process of claim 17, wherein theprocess is performed at a temperature in the range of about 20° C. toabout 40° C.
 20. The process of claim 17, further comprising stirringthe mixture for at least 2 hours.